Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy.

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. This chapter describes a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.

Structure-Guided Design of a Peptide Lock for Modular Peptide Binders.

Peptides play an important role in intermolecular interactions and are frequent analytes in diagnostic assays, also as unstructured, linear epitopes in whole proteins. Yet, due to the many different sequence possibilities even for short peptides, classical selection of binding proteins from a library, one at a time, is not scalable to proteomes. However, moving away from selection to a rational assembly of preselected modules binding to predefined linear epitopes would split the problem into smaller parts. These modules could then be reassembled in any desired order to bind to, in principle, arbitrary sequences, thereby circumventing any new rounds of selection. Designed Armadillo repeat proteins (dArmRPs) are modular, and they do bind elongated peptides in a modular way. Their consensus sequence carries pockets that prefer arginine and lysine. In our quest to select pockets for all amino acid side chains, we had discovered that repetitive sequences can lead to register shifts and peptide flipping during selections from libraries, hindering the selection of new binding specificities. To solve this problem, we now created an orthogonal binding specificity by a combination of grafting from ß-catenin, computational design and mutual optimization of the pocket and the bound peptide. We have confirmed the design and the desired interactions by X-ray structure determination. Furthermore, we could confirm the absence of sliding in solution by a single-molecule Förster resonance energy transfer. The new pocket could be moved from the N-terminus of the protein to the middle, retaining its properties, further underlining the modularity of the system.

Charge Interactions Can Dominate Coupled Folding and Binding on the Ribosome.

Interactions between emerging nascent polypeptide chains and the ribosome can modulate cotranslational protein folding. However, it has remained unclear how such interactions can affect the binding of nascent chains to their cellular targets. We thus investigated on the ribosome the interaction between two intrinsically disordered proteins of opposite charge, ACTR and NCBD, which form a high-affinity complex in a coupled folding-and-binding reaction. Using fluorescence correlation spectroscopy and arrest-peptide-mediated force measurements in vitro and in vivo, we find that the ACTR-NCBD complex can form cotranslationally but only with ACTR as the nascent chain and NCBD free in solution, not vice versa. We show that this surprising asymmetry in behavior is caused by pronounced charge interactions: attraction of the positively charged nascent chain of NCBD to the negatively charged ribosomal surface competes with complex formation and prevents ACTR binding. In contrast, the negatively charged nascent ACTR is repelled by the ribosomal surface and thus remains available for productively binding its partner. Electrostatic interactions may thus be more important for cotranslational folding and binding than previously thought.

Internal friction in an intrinsically disordered protein-Comparing Rouse-like models with experiments.

Internal friction is frequently found in protein dynamics. Its molecular origin however is difficult to conceptualize. Even unfolded and intrinsically disordered polypeptide chains exhibit signs of internal friction despite their enormous solvent accessibility. Here, we compare four polymer theories of internal friction with experimental results on the intrinsically disordered protein ACTR (activator of thyroid hormone receptor). Using nanosecond fluorescence correlation spectroscopy combined with single-molecule Förster resonance energy transfer (smFRET), we determine the time scales of the diffusive chain dynamics of ACTR at different solvent viscosities and varying degrees of compaction. Despite pronounced differences between the theories, we find that all models can capture the experimental viscosity-dependence of the chain relaxation time. In contrast, the observed slowdown upon chain collapse of ACTR is not captured by any of the theories and a mechanistic link between chain dimension and internal friction is still missing, implying that the current theories are incomplete. In addition, a discrepancy between early results on homopolymer solutions and recent single-molecule experiments on unfolded and disordered proteins suggests that internal friction is likely to be a composite phenomenon caused by a variety of processes.

The Three-Fold Axis of the HIV-1 Capsid Lattice Is the Species-Specific Binding Interface for TRIM5α.

Rhesus TRIM5α (rhTRIM5α) potently restricts replication of human immunodeficiency virus type 1 (HIV-1). Restriction is mediated through direct binding of the C-terminal B30.2 domain of TRIM5α to the assembled HIV-1 capsid core. This host-pathogen interaction involves multiple capsid molecules within the hexagonal HIV-1 capsid lattice. However, the molecular details of this interaction and the precise site at which the B30.2 domain binds remain largely unknown. The human orthologue of TRIM5α (hsTRIM5α) fails to block infection by HIV-1 both in vivo and in vitro This is thought to be due to differences in binding to the capsid lattice. To map the species-specific binding surface on the HIV-1 capsid lattice, we used microscale thermophoresis and dual-focus fluorescence correlation spectroscopy to measure binding affinity of rhesus and human TRIM5α B30.2 domains to a series of HIV-1 capsid variants that mimic distinct capsid arrangements at each of the symmetry axes of the HIV-1 capsid lattice. These surrogates include previously characterized capsid oligomers, as well as a novel chemically cross-linked capsid trimer that contains cysteine substitutions near the 3-fold axis of symmetry. The results demonstrate that TRIM5α binding involves multiple capsid molecules along the 2-fold and 3-fold interfaces between hexamers and indicate that the binding interface at the 3-fold axis contributes to the well-established differences in restriction potency between TRIM5α orthologues.IMPORTANCE TRIM5α is a cellular protein that fends off infection by retroviruses through binding to the viruses' protein shell surrounding its genetic material. This shell is composed of several hundred capsid proteins arranged in a honeycomb-like hexagonal pattern that is conserved across retroviruses. By binding to the complex lattice formed by multiple capsid proteins, rather than to a single capsid monomer, TRIM5α restriction activity persists despite the high mutation rate in retroviruses such as HIV-1. In rhesus monkeys, but not in humans, TRIM5α confers resistance to HIV-1. By measuring the binding of human and rhesus TRIM5α to a series of engineered HIV-1 capsid mimics of distinct capsid lattice interfaces, we reveal the HIV-1 capsid surface critical for species-specific binding by TRIM5α.

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins.

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules.

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts.

The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.

Intramolecular distances and dynamics from the combined photon statistics of single-molecule FRET and photoinduced electron transfer.

Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution.